Abstract
OBJECTIVES: To investigate the mechanism of eurycomanone (EN) for ameliorating ischemia/reperfusion (IR)-induced acute kidney injury (AKI) in mice. METHODS: Twenty-four male C57BL/6J mice were randomly divided into 4 groups (n=6) for sham operation, IR modeling by bilateral renal pedicle clamping, or intraperitoneal injections of EN at 0.25 or 1.0 mg/kg for two days prior to surgery. Network pharmacology and molecular docking were used to identify the potential molecular targets and signaling pathways and evaluate the binding affinities. Renal function, histopathological changes, expression of tubular injury markers (KIM-1 and NGAL), phosphorylation levels of STAT3, PI3K, and JAK2, renal inflammation, mitochondrial biogenesis, and mitochondrial function of the mice were assessed. The interaction between EN and STAT3 was examined using surface plasmon resonance (SPR). Another 24 male C57BL/6J mice receiving sham operation, IR modeling, or EN or EN+ML115 (a STAT3 agonist) treatment prior to modeling (n=6) were used to validate the role of STAT3 in EN-mediated renal protection. RESULTS: Both low- and high-dose EN significantly alleviated renal injury and improved renal function in mice with IR-induced AKI. The core targets of EN were associated with inflammation-related signaling pathways. EN treatment markedly reduced renal levels of IL-6, MCP-1, and TNF-αand decreased macrophage infiltration (F4/80-positive cells) in renal interstitial tissue of the mice. EN also significantly increased renal ATP content and mitochondrial DNA copy number (P<0.05), and upregulated the expressions of PGC-1α, TFAM, and Nrf2 mRNAs and protein levels of PGC-1α and TOM20. Molecular docking identified STAT3 and PI3K as key molecular targets of EN. In mice with IR-induced AKI, EN significantly suppressed phosphorylation of STAT3 and PI3K in the renal tissues without affecting p-JAK2 levels. SPR analysis confirmed a direct and specific interaction between EN and STAT3. Notably, activation of STAT3 by ML115 significantly reversed the renoprotective effects of EN. CONCLUSIONS: EN mitigates IR-induced AKI in mice by directly binding to STAT3 to inhibit its phosphorylation, suppressing inflammation, and enhancing mitochondrial biogenesis and function, suggesting the potential of EN as a promising therapeutic candidate for IR-induced AKI.